The overall aim of this proposal is to better understand the cellular and molecular underpinnings of terminal erythroid cell maturation. Our red cell mass is sustained by a robust process of cell maturation that is primarily dependent on the hormone erythropoietin (EPO). EPO, signaling through its cognate receptor (EPOR) provides a critical survival signal to late-stage definitive erythroid progenitors making it difficult to study its role in the downstream terminal maturation o erythroid precursors. While EPO is widely recognized to be essential for definitive erythropoiesis, its role in primitive erythropoiesis, a transient lineage necessary for embryonic growth and survival, remains controversial and poorly understood. We hypothesize that EPO plays a central role in the terminal maturation of the primitive erythroid lineage. This hypothesis will be tested in Aim 1 studies by delineating the role of EPO in mouse embryos lacking EPOR as well as in an ex vivo 2-step primitive erythroid culture system. Our recent studies of radiation injury to the bone marrow have led us to hypothesize that terminal erythroid cell maturation is characterized by a transition from a pro-apoptotic to an anti-apoptotic state (Peslak, 2011). Preliminary studies indicate that EPO prevents the apoptosis of late-stage primitive erythroid precursors. The persistence of mutant primitive erythroid cells in EPOR-null embryos provides a unique opportunity to investigate the role of EPO in terminal stages of erythroid cell maturation. We have identified pro- and anti-apoptotic genes whose levels are dramatically altered by loss of EPO. In Aim 2, we will more fully characterize the expression of pro- and anti-apoptotic genes in wild-type and EPOR-null erythroblasts. Furthermore, we will delineate the role of Stat signaling in the regulation of Bcl-xL both in wild- type and in mutant primitive erythroblasts lacking Stat5 signaling. Our studies of EPO function in late stages of erythroid maturation may ultimately provide insights, by comparison, into the role of this clinically important cytokine in EPO-responsive non-erythroid cells, both normal and malignant. (End of Abstract)